阅读说明：本技术 一种高纯度酶改制甜菊糖苷及其制备方法 (High-purity enzyme-modified stevioside and preparation method thereof ) 是由 周爱琴 周爱兵 于茂兰 张璐璐 于 2020-08-06 设计创作，主要内容包括：本发明属于甜菊糖技术领域,提供了一种高纯度酶改制甜菊糖苷的制备方法,包括：将甜菊糖苷与糊精溶解在水中；添加反应酶,搅拌均匀；在60-70℃条件下反应24-48h；添加糊精继续反应5h,添加量为糊精量的10％-20％；升温至90-105℃将酶灭活；冷却后,活性炭过滤,除去颜色和杂质；将溶液配制成合适浓度,过大孔吸附树脂,至溶液吸附完全,再用2倍吸附柱体积的水冲洗吸附柱；用4倍吸附柱体积的40％-50％的乙醇溶液洗脱,将大孔吸附树脂树脂间的未反应的甜菊糖苷和酶改制甜菊糖苷混合物洗脱下来；流出液紫外吸光度大于0.1-0.3时进行收集1-1.5BV的流出液；将流出液浓缩后喷干,获得酶改制甜菊糖苷。(The invention belongs to the technical field of stevioside, and provides a preparation method of high-purity enzyme-modified stevioside, which comprises the following steps: dissolving stevioside and dextrin in water; adding reaction enzyme, and stirring uniformly; reacting for 24-48h at 60-70 ℃; adding dextrin to continue reacting for 5h, wherein the addition amount is 10% -20% of the paste amount; heating to 90-105 deg.C to inactivate enzyme; after cooling, filtering with activated carbon to remove color and impurities; preparing the solution into a proper concentration, passing through macroporous adsorption resin until the solution is completely adsorbed, and washing the adsorption column by using 2 times of the volume of the adsorption column; eluting with 40% -50% ethanol solution 4 times the volume of the adsorption column, and eluting unreacted stevioside and enzyme-modified stevioside mixture between macroporous adsorbent resin; collecting the effluent of 1-1.5BV when the ultraviolet absorbance of the effluent is more than 0.1-0.3; concentrating the effluent, and spray drying to obtain enzyme-modified stevioside.)

1. A preparation method of high-purity enzyme-modified stevioside is characterized by comprising the following steps:

step S1: dissolving stevioside and dextrin in water;

step S2: adding reaction enzyme into the solution of the step S1, and stirring uniformly;

step S3: reacting the solution obtained in the step S2 at 60-70 ℃ for 24-48 h;

step S4: after the reaction in the step S3 is finished, adding dextrin to continue the reaction for 5 hours, wherein the adding amount is 10% -20% of the paste amount in the step S1;

step S5: heating the reactant in the step S4 to 90-105 ℃, and inactivating the enzyme;

step S6: after cooling, the reaction solution of step S5 is filtered with activated carbon to remove color and impurities;

step S7: preparing the solution obtained in the step S6 into a solution with the mass concentration of 1% -5%, passing through macroporous adsorption resin until the solution is completely adsorbed, wherein the flow rate is 1BV/h, washing the adsorption column by using 2 times of the volume of the adsorption column, and washing away residual dextrin;

step S8: eluting with 40% -50% ethanol solution 4 times the volume of the adsorption column, eluting the unreacted stevioside and enzyme-modified stevioside mixture between macroporous adsorption resin resins at a flow rate of 0.5 BV/h; collecting the effluent liquid with the volume of 1-1.5BV when the ultraviolet absorbance of the effluent liquid is more than 0.1-0.3;

step S9: concentrating the effluent, and spray drying to obtain enzyme-modified stevioside.

2. The method according to claim 1, wherein in step S1, the mass ratio of steviol glycoside to dextrin is 1: 0.5-3, and the mass concentration of stevioside is 1-20%.

3. The method according to claim 1, wherein in step S2, the amount of the reaction enzyme is 1-5% by mass of the steviol glycoside.

4. The method of claim 1, wherein the reaction solution is stirred at a constant speed of 50-100rpm/min during the reaction in step S3.

5. The method of claim 1, wherein the dextrin is cyclodextrin or maltodextrin, or starch.

6. The method of claim 1, wherein the dextrin has a DE value of 10 to 15.

7. The method of claim 1, wherein the steviol glycoside has a purity of at least 80%.

8. The method for preparing stevioside by enzyme modification with high purity according to claim 1, wherein the reaction enzyme is cyclodextrin glucosyltransferase or a lyase formed by cyclodextrin glucosyltransferase and alpha-amylase in a mass ratio of 1:1.

9. The method according to claim 1, wherein in step S9, the step of concentrating the effluent and then drying the concentrated effluent is to concentrate the effluent into a feed liquid with a mass concentration of 40% -50%, and then to perform spray drying, wherein the feed is performed when the vacuum degree in the spray dryer reaches-95 kpa, the feed rate is 120kg/h, the air inlet temperature is 195 ℃, and the air outlet temperature is 95 ℃.

10. The method of making a high purity enzyme-modified steviol glycoside according to any one of claims 1 to 9, wherein the enzyme-modified steviol glycoside has a purity of at least 80%.

Technical Field

The invention relates to the technical field of stevioside, and in particular relates to stevioside prepared by modifying high-purity enzyme and a preparation method thereof.

Background

Stevioside (also called stevioside) is a mixture of multicomponent sweet glycosides extracted from the leaves of the natural herb stevia rebaudiana Bertoni of Compositae, is the main flavor substance in stevia rebaudiana and is used as a food sweetener in production. Stevioside mainly comprises eight kinds of glycosides such as stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, dulcoside, steviolbioside and the like. The stevioside has the advantages of pure nature (from pure natural plant stevia rebaudiana), high sweetness (250-450 times of cane sugar), low calorie (only 1/300 of white sugar), economic use (the cost is only one third of that of cane sugar), good stability (heat resistance, acid resistance, alkali resistance, difficult decomposition phenomenon and no toxic or side effect), high safety and the like.

However, the structural glycoside of natural stevioside has no sweetness and has bitter taste, wherein stevioside is one of the components with the highest content in stevioside, the afterbitterness is obvious, the mouthfeel of the stevioside is damaged, and the wider industrial application of the stevioside is limited. Usually, enzyme modification of steviol glycosides is carried out, i.e. one or more molecules of glucose are added randomly to the steviol glycoside molecule by cyclodextrin glucose transferase.

The existing enzyme modification technology is complex in operation, and finally obtained enzyme-modified stevioside has different purities and lower content, so that the application of the enzyme-modified stevioside is limited.

Disclosure of Invention

It is an object of the present application to overcome the above problems or to at least partially solve or mitigate the above problems.

In a first aspect, the present invention provides a method for preparing a steviol glycoside modified with a high-purity enzyme, comprising the steps of:

step S1: dissolving stevioside and dextrin in water;

step S2: adding reaction enzyme into the solution of the step S1, and stirring uniformly;

step S3: reacting the solution obtained in the step S2 at 60-70 ℃ for 24-48 h;

step S4: after the reaction in the step S3 is finished, adding dextrin to continue the reaction for 5 hours, wherein the adding amount is 10% -20% of the paste amount in the step S1;

step S5: heating the reactant in the step S4 to 90-105 ℃, and inactivating the enzyme;

step S6: after cooling, the reaction solution of step S5 is filtered with activated carbon to remove color and impurities;

step S7: preparing the solution obtained in the step S6 into a solution with the mass concentration of 1% -5%, passing through macroporous adsorption resin until the solution is completely adsorbed, wherein the flow rate is 1BV/h, washing the adsorption column by using 2 times of the volume of the adsorption column, and washing away residual dextrin;

step S8: eluting with 40% -50% ethanol solution 4 times the volume of the adsorption column, and eluting the unreacted stevioside and enzyme-modified stevioside mixture between macroporous adsorption resin resins at flow rate of 0.5 BV/h; collecting the effluent liquid with the volume of 1-1.5BV when the ultraviolet absorbance of the effluent liquid is more than 0.1-0.3;

step S9: concentrating the effluent, and spray drying to obtain enzyme-modified stevioside.

This application detects absorbance through using ultraviolet analyzer to effluent, and absorbance is greater than 0.1-0.3 and shows that enzyme reforms stevioside and flows out.

Optionally, in step S1, the mass ratio of steviol glycoside to dextrin is 1: 0.5-3, and the mass concentration of stevioside is 1-20%.

Optionally, in the step S2, the amount of the reactive enzyme added is 1% to 5% of the mass of the steviol glycoside.

Optionally, in step S3, the reaction solution is stirred at a constant speed during the reaction, and the rotation speed is 50-100 rpm/min.

Optionally, the dextrin is cyclodextrin or maltodextrin, or replaced by starch.

Optionally, the dextrin has a DE value of 10 to 15.

Optionally, the steviol glycoside has a purity of at least 80%.

Optionally, the reaction enzyme is cyclodextrin glucosyltransferase or a combined enzyme formed by cyclodextrin glucosyltransferase and alpha-amylase according to the mass ratio of 1:1.

Optionally, in step S9, the step of concentrating the effluent and then spray-drying the concentrated effluent is to concentrate the effluent into a feed liquid with a mass concentration of 40% to 50%, and then spray-drying the feed liquid, wherein the feed liquid is fed when the vacuum degree in the spray-drying machine reaches-95 kpa, the feed speed is 120kg/h, the temperature of the air inlet is 195 ℃, and the temperature of the air outlet is 95 ℃.

In a second aspect, the present invention provides an enzyme-modified steviol glycoside prepared according to the method for preparing a high-purity enzyme-modified steviol glycoside, wherein the purity of the enzyme-modified steviol glycoside is at least 80%.

The invention has the beneficial effects that:

1. according to the preparation method of the high-purity enzyme-modified stevioside, the glucosyl group is added on the structure of the stevioside, the enzyme-modified stevioside with higher purity can be prepared, the purity is at least 80 percent and can reach 98 percent at most, the unreacted dextrin is almost zero, the sweet taste is natural and pure, the natural fruit fragrance is provided, the unpleasant aftertaste of the stevioside is completely avoided, the color is pure white, the solubility is greatly improved, and the application range of the enzyme-modified stevioside is further expanded.

2. The prepared high-purity enzyme-modified stevioside is added into stevioside mother liquor sugar, the taste of the mother liquor sugar is greatly improved, and the sweetness is also increased.

3. This application utilizes the principle that steviol glycoside and enzyme change stevioside structure are different, utilizes the ethanol elution of specific concentration, and enzyme change stevioside and wherein a small part unreacted steviol glycoside can flow out preferentially, and other most unreacted steviol glycoside and a small part enzyme change stevioside and elute at last.

3. The preparation method is simple to operate and suitable for industrial production.

Drawings

In order to more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings that are needed in the detailed description of the invention or the prior art will be briefly described below. Throughout the drawings, like elements or portions are generally identified by like reference numerals. In the drawings, elements or portions are not necessarily drawn to scale.

FIG. 1 is a basic structural formula of steviol glycosides;

fig. 2 shows a flow chart of a preparation method of enzyme-modified steviol glycoside provided in the embodiment of the invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.

It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.

Figure 1 is the structural formula of steviol glycoside. All components of stevioside contain the same aglycone (Steviol ), and because different amounts of glucosyl or rhamnosyl are connected to positions C19 and C13, the sweetness and taste quality of the stevioside are greatly different. It is generally believed that products having various sugar groups attached to the C13 position may have better sweetness and taste profile. The essence of enzyme-modified stevioside is that glucosyl is added on the main structure C19 or C13 of stevioside, and the sweetness and taste quality of stevioside are changed.